Preparation Of Anti-human CD105Antibody And Its Preliminary Application On Imaging And Detection Of Ovarian Cancer

一、Background：Ovarian cancer is the leading cause of death from gynecological cancers. Primarytreatment of advanced ovarian cancer until recently consisted of cytoreductive surgery andpaclitaxel/carboplatin chemotherapy. Unfortunately, most patients died of drug resistanceand recurrence. Angiogenesis is essential for oncogenesis but also the viability andexpansion of ovarian cancer. Antiangiogenic therapy is promising to control and cureovarian cancer. The results of two randomized studies, showing prolongation ofprogression-free survival （PFS） by the addition of the anti-VEGF monoclonal antibody,bevacizumab, led to the approval of this agent for first-line treatment of this disease andindicate that angiogenesis is a promising therapeutic target.Endoglin, also known as CD105, is one such antigen gaining widespread popularity. Itis classified as an accessory receptor for transforming growth factor beta （TGF-β）, apleiotropic cytokine regulating cellular proliferation, differentiation, migration and adhesion.Remarkably, its expression is upregulated in actively proliferating endothelial cells.Endoglin has therefore been suggested as an appropriate marker for tumor-relatedangiogenesis and neovascularization.High levels of endoglin expression on actively proliferating tumoral endothelial cellson the luminal surface made CD105an ideal target to the diagnosis and therapy of ovariancancer. Plenty of studies have proved that targeting CD105made it possible to imagine andtreat xenografts of experimental animals. The soluble form of the receptor generated viaMMP-14-dependent cleavage of membrane-bound endoglin has been evaluated forprognostic purposes. Several researchers have reported increased levels of sEndoglin inserum of pregnant women diagnosed with pre-eclampsia. sEndoglin has been correlatedwith metastatic disease in breast and colorectal cancer patients.Ovarian cancer is a highly vascularized tumor, anti-human CD105antibodies may play an important role in the diagnosis and treatment of ovarian cancer. Most Study ofanti-CD105antibody focused on the monoclonal antibody （McAb）. CD105-McAb havebeen merchandised in foreign countries, but no functional anti-CD105antibody has beenreported inland. It is of great significance to develop CD105-McAb for clinical diagnosisand treatment with independent intellectual property rights.Preparation of anti-human CD105antibodies can be applied not only on anti-tumordrug screening, but also to build immunoassay technology for metastasis and prognosisevaluation of ovarian cancer. However, preparation of an antibody drug is difficult, in termsof high requirements of specificity, affinity, blocking activity and biological reactivity.However, diagnostic applications of McAb is relatively easy. Therefore，after successfulpreparation of anti-human CD105antibody, we can take advantage of the specificity ofCD105antibody to establish measures to detect CD105in vivo and in vitro.Since the1970s with the putting forward theories of the detection based onelectrochemical immunosensor, electrochemical immunosensor which is combined with theelectrochemical method with high sensitivity and stability and immunoassay with highselectivity has been widely used in the medical, biological detection. Furthermore, it hasreceived more and more attentions. Electrochemical immunosensor is with similarsensitivity of radioimmunoassay, but without the using of radioisotopes, so there is noradiation injury for the user of medical personnel and patients, and no damage for theantibody or antigen when marked with radio chemical, which could retain the maximumbio-activity of them to improve the detection efficiency. These studies indicate the goodapplication and development prospects of electrochemical immunosensor, but there aresome limit of the study and using of it because of the technology limit of protein expressionand purification and preparation of antibodies, especially monoclonal antibodies with hightiters. Because of these limits, most of the immunoassay products were dependent onimports, especially for high-end products. It’s important and urgent to improve thetechnology for protein expression and purification and preparation of antibodies with hightiters, and then develop novel electrochemical immunosensor with high sensitivity,selectivity and stability, which could be used for the application in clinical and biologicaldetection and confirm the application of studied antigens and antibodies. These studies willbe important with scientific significance, social and economic value. 二、Methods and results：1.Molecular Cloning of extracellular region of human CD105and construction ofhighly efficient prokaryotic expression vector and protein purification system:Extracellular fragment of CD105molecule was cloned by PCR. Restriction enzymedigestion and sequence analysis showed that its sequence completely consistent with theliterature. This fragment was subcloned into the expression vector pET28a （+）, transformedto BL21. After induced by IPTG, expressing protein was analyzed by SDS-PAGE, whichshowed a distinct zone at the place of60Dk with its molecular size in line with expectations.Western-blot analysis showed that the expressed protein band could be recognized speciallyby commercialized CD105-McAb. Expression products presents with the form of inclusionbodies, purified with Ni+-nitrilotriacetic acid （Ni+-NTA） affinity chromatogram, refoldedby dialysis. At last, we obtained recombinant CD105protein with purity up to90%.2.Preparation and identification of anti human CD105monoclonal antibody:2.1Preparation of CD105linear polypeptide epitope-specific polyclonal antibodyIn order to obtain more CD105McAb targeting different epitopes, to create aDouble-antibody sandwich enzyme-linked immunosorbent assay （ELISA） detection ofCD105,we designed and synthesized two epitope polypeptides and conjugated it with BSA.The rabbit was immunized with the polypeptide conjugated to BSA, high titer antibody wasobtained.Purified antibodies were tested by ELISA, SDS-PAGE and proved to be CD105specificity.2.2Preparation and characterization of anti-CD105monoclonal antibodyUsing self-made purified CD105extracellular proteins as immunogen, we adopt amodified immunization program and classic hybridoma technology. After experienced fourcycles of cell fusion, we screened six strains of anti-glycosylased CD105monoclonalantibodies from dozens clones of positive strains. Specificity of anti-CD105McAb wasdemonstrated by ELISA, Western-Blot, immunofluorescence.3.131-iodine labeled anti-CD105McAb and tumor radioimmunoimaging （RII）innude mice bearing human ovarian cancer:3.1Iodogen method was used for preparing¹³¹I labeling CD105monoclonal antibodyand control monoclonal antibody（mouse anti human antibody,mIgG）. SephadexG50columnwas used to purify the product. Labeling efficiency was calculated and the radiochemical purity was measured by using paper chromatography method. The labeling rate of¹³¹I toCD105is92.2%and83.6%respectively，and the radiochemistry purity is more than97%.3.2After the nude mice were injected with¹³¹I-CD105and¹³¹I-mIgG through tailveil, mice were sacrificed at different time points. The interested organs were excised,washed with saline, weighed, and counted on a gamma counter for calculating dose pergram of tissue （%ID/g）, tumor/non-tumor （T/NT） ratio. Immunoradioimagining of thetumors was acquired using SPECT at24h，48h，96h after injection.Experimental group has a higher tissue uptake of the percentage injected （%ID/g） inheart, liver, lung, kidney, stomach24h after injection. With time goes on, the rate oftumor tissue uptake increased significantly, while other important organ uptake ratedecreased gradually. At96hours after injection,%ID/g of tumor increased to29.10±1.61,while the liver, kidney and other organs decreased significantly. Corresponding T/NT ratioreached more than3at96hours after injection, which proved that¹³¹I-CD105monoclonalantibody concentrates at the tumor site and reached a high point in96hours.While incontrol group, we failed to find radioactivity accumulation at tumor site.Significant radioactivity accumulation was observed in the tumor site, but alsoaccumulated in kidney, liver, and stomach24h after injection.As time goes on, the contourof xenograft can be sharply displayed at48h after injection.At96h, tumor outline becamedistinct while background faded.4. Study of electrochemical immunosensor for the detection of human CD105Based on the preparation of antigen and antibody, we developed a novelelectrochemical immunosesnor for the detection of human CD105as followed. Firstly, weprepared a monolayer of gold nanoparticles with good biological compatibility to improvethe surface area and amount of modified antibodies. To improve the surface area more,mercaptoethylamine was modified onto the electrode surface to adsorb gold nanoparticleswith lower particle size. And then, monoclonal antibody for human CD105was modifiedonto the electrode surface by the interaction between the antibody and gold nanoparticle todevelop the electrochemical immunosensor for the detection of human CD105. At the sametime, we used platinum nanoparticles with catalytic ability for peroxide and thionine as anelectrochemical mediator to be marked onto the second antibody to enhance theelectrochemical reaction signals and detection limit. At the same time, using of second antibody will improve the selectivity of immunoassay. In this study, cyclic voltammetry wasused for the preparation and characterization of the electrochemical immunosensor. Itshowed satisfactory result in the application study which indicates good practicality of thestudied electrochemical immunosesnor and satisfactory application performance of theprepared antigen and antibody with good possible application. Furthermore, these studieswill make a well technological foundation to develop and improve new clinical detectionmethod and other possible application.